Heterogeneous TCR delta Vdelta2-Ddelta3 rearrangements and their relation to IgH and TCR gamma gene status in childhood B cell precursor leukaemias.

In this study we aimed to test the hypothesis that leukaemias with oligoclonal Vdelta2-Ddelta3 rearrangements are clonal at the IgH and TCRG gene status and that the oligoclonality is a poor prognostic marker. In ten leukaemias the individual Vdelta2-Ddelta3 rearrangements characterised different populations as deduced from single cell analysis and/or from densitometric differences of PCR products. Five of these leukaemias were clonal by the IgH and/or TCRG gene status. We therefore conclude that ALL is a clonal disease despite the presence of heterogeneous TCRD rearrangements. Our clinical data show that oligoclonality at the TCRD level does not represent an adverse prognostic factor.

Ewing tumor after treatment of Ki-1+ anaplastic large cell lymphoma. Therapy-associated secondary neoplasm or unrelated coincidence?

We report on a 20-year-old woman who developed a pelvic small round cell tumor with lung metastases 8 years after diagnosis and successful treatment for Ki-1-positive anaplastic large cell lymphoma (Ki-1+ ALCL) with histiocytic differentiation. Molecular genetic detection of EWS/FLI-1 fusion gene expression in the second tumor by RT-PCR unambiguously confirmed the histopathologic diagnosis of Ewing tumor (ET), whereas no evidence for the presence of this specific gene rearrangement was obtained in a retrospective analysis of the lymphoma tissue. In contrast, expression of a NPM/ALK chimeric gene was observed which was absent in the ET. Moreover, the lymphoma contained a monoallelic D delta 2-D delta 3 T-cell receptor gene rearrangement which was also absent in the ET. Thus, our histopathologic, immunohistochemical, and, in particular, molecular genetic studies support the notion that these tumors were most probably pathogenetically unrelated. Since this is the first report describing such an association between a non-Hodgkin's lymphoma and ET and, since the latter has only rarely been observed as a second malignant neoplasm, it remains a matter of speculation whether in this patient ET developed as a therapy-related secondary neoplasm or independently from the lymphoma as a consequence of either genetic tumor predisposition or mere accidental coincidence.

Subcellular compartmentation of penicillin biosynthesis in Penicillium chrysogenum. The amino acid precursors are derived from the vacuole.

The cellular localization of the origin of alpha-aminoadipate used in penicillin biosynthesis and the first enzymic step in Penicillium chrysogenum involved, delta-(alpha-aminoadipyl)-L-cysteinyl-D-valine synthetase (ACVS), has been studied. Subcellular fractions were obtained from protoplasts of a high penicillin-producing strain upon lysis by Triton X-100, and vacuoles purified from them. They were identified by the aid of alpha-mannosidase as a marker enzyme, by the presence of polyphosphate, and their ability to sequester [14C]lysin, added to the protoplasts prior to subcellular fractionation. 15.6 and 26.5%, respectively, of 6-[14C]alpha-aminoadipate, and 8.5 and 10.3%, respectively, of [14C]valine added accordingly were also found in the vacuole, and the higher proportion was found in vacuoles isolated from penicillin-producing mycelia. ACVS protein was detected in the membrane as well as the soluble fraction of the purified vacuoles. We propose therefore that ACVS is located either within or bound to the vacuolar membrane, and that the precursor amino acids for penicillin biosynthesis are withdrawn from the vacuolar amino acid pool.

A rare leucine 40/arginine40 polymorphism on platelet glycoprotein IIIa is linked to the human platelet antigen 1b.

A rare polymorphism on glycoprotein (GP) IIIa is reported. Sequencing of a 482-base-pair (bp) PCR product of the genomic DNA of GPIIIa revealed a single-base exchange of a G<==>T polymorphism at base 12,569 that created an additional restriction site for MspI. This single-point mutation (frequency in Caucasians 0.00386) is on the HPA-1b gene (HPA-1bvar) and codominantly inherited. The exchange of a G for a T results in a leucine-to-arginine substitution at amino acid 40 from the NH2 terminus. The binding of anti-HPA-1a and -1b antibodies to HPA-1bvar platelets is not influenced.

NPM/ALK gene fusion transcripts identify a distinct subgroup of null type Ki-1 positive anaplastic large cell lymphomas.

The chromosomal aberration t(2:5) resulting in the juxtaposition of NPM and ALK genes is a well-known feature of several Ki-1+ anaplastic large cell lymphomas (ALCL) of the T-cell type. However, conflicting results have been reported concerning the presence of this gene rearrangement in other ALCL and Hodgkin's disease (HD), respectively. We performed NPM/ALK RT-PCR on 14 cases of ALCL expressing distinct myelomonocytic markers, e.g. CD11c, CD13, CD14 or CD68, but neither T-cell nor B-cell associated antigens (null cell phenotype). The specific translocation was found exclusively in six childhood tumours previously diagnosed as malignant histiocytosis (MH), whereas all adult lymphomas (three ALCL without characteristics of MH, three secondary ALCL following HD) and two paediatric cases of secondary ALCL following HD did not show NPM/ALK gene fusion products. By Southern blotting, the status of T-cell receptor (TCR) and immunoglobulin heavy chain genes (IgH) were investigated; two patients with initially diagnosed MH had the TCRdelta-chain gene rearranged (Ddelta2-Ddelta3 and Vdelta1-Jdelta1, respectively). IgH rearrangements were detected in only one patient with secondary ALCL. Our data indicate a high association of previously diagnosed MH and NPM/ALK gene rearrangements. In one case, this specific translocation was demonstrated at an early stage of development; in another, a mature TCRdelta-chain gene rearrangement was detected. These data support the hypothesis of a lymphoid origin of this subgroup of Ki-1 positive ALCL previously diagnosed as MH.

Heterogeneity of the T-cell receptor delta gene indicating subclone formation in acute precursor B-cell leukemias.

Precursor B-cell acute lymphoblastic leukemias (B-ALLs) have been shown to be oligoclonal at the Ig heavy-chain (IgH) gene level in up to 40% of cases by Southern blot hybridization. In contrast, oligoclonality as deduced from diversity of T-cell receptor (TcR)-delta gene rearrangements of the immature types (ie, V delta 2-D delta 3, D delta 2-D delta 3) has not been reported, so far. We detected oligoclonality characterized by the coexistence of different junctional regions of identical V delta 2-D delta 3 rearrangements in four childhood precursor B-ALLs. No variation was found in the IgH gene status. Therefore, we define these populations as subclones. Two leukemias displayed the variants in an unequal proportion. In the other two leukemias, for which similar quantities of the coexisting rearrangements were detected, single cell-nuclei polymerase chain reaction (PCR) showed two separate leukemic populations. Subclone formation could not be demonstrated by Southern blot hybridization, but was detectable after PCR amplification of the V delta 2-D delta 3 rearrangement and separation by polyacrylamide gel electrophoresis. The variants arose independently from each other, as deduced from their individual sequences. Using subclone-specific oligonucleotides for hybridization to amplified DNA obtained at diagnosis and during follow-up from bone marrow samples, we demonstrate, (1) specificity of all subclone-deduced probes, (2) that one residual leukemic cell can be detected in 10(4) to 10(5) normal mononuclear cells in a semiquantitative assay, and (3) that none of the subclones persisted after induction therapy. We propose that in a leukemic cell population, TcR-delta gene diversity arises after rearrangements of the IgH genes resulting in apparent clonality at the IgH gene level. However, cells are oligoclonal, if the TcR-delta gene rearrangements are considered. As various subclones may respond differently to chemotherapy, they may hamper the detection of minimal residual disease. Therefore, we use all subclone-specific oligonucleotides for hybridization to amplified DNA from follow-up samples.